Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's. Heavily implicated in its molecular pathology is the abundantly-expressed and intrinsically disordered protein (IDP) a- synuclein (a -syn). While the precise role played by a -syn in PD is unknown, its aggregation into filamentous inclusion bodies within the brain is the defining hallmark of this and other synucleinopathies. Understanding the aggregation process of a -syn and its connection to PD is thus of great interest to the both academic and medical research communities as such information is anticipated to contribute greatly to development of effective therapies, of which there are currently none. Despite much effort however, such molecular-level information remains elusive. IDPs are defined in part by their lack of well-defined structure in vitro, apparently relying on functionally relevant binding partners to modulate and affect conformations in vivo. This characteristic structural plasticity makes IDPs inherently difficult to study as the effects of conformational averaging of conventional structural studies often lead to a dramatic loss of information about sub-populations and conformational heterogeneity critical to understanding folding in these anomalous proteins. In the case of a-syn, this problem is further complicated by an expansive range of known binding partners (small molecules, metal ions, and other proteins) of unknown biological relevance, and presently unknown cellular function(s). To overcome these challenges and reveal otherwise hidden information about the structure of a-syn critical to understanding its role and function(s), single-molecule fluorescence (SMF) methods such as Forster Resonance Energy Transfer (smFRET) in combination with Fluorescence Correlation Spectroscopy (FCS, auto- and cross-correlation) are proposed herein to probe this challenging target. The Deniz lab is a world leader in the application of these highly sensitive techniques, which report directly on even complex populations through observation structural features of individual molecules. These SMF methods will be applied to comprehensively profile and model the structural properties of a-syn by (i) examining in detail the behavior of monomeric a-syn under both non-aggregating and aggregation-promoting conditions, including a focus on the C-terminal "tail" region, hypothesized to mitigate aggregation but which has been largely neglected due to a high level of disorder in solution (presents a significant challenge for non-SMF studies), and (ii) characterizing the global structural rearrangements that a-syn undergoes in the presence of several known binding partners of suspected biological relevance. It is anticipated that such detailed information about molecular conformation and dynamics will allow for the development of a detailed structural model of a-syn function in normal and diseased states, and ultimately for a better understanding IDPs and their broad role in human health. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, and has devastating effects on the muscular and cognitive abilities of those who suffer from it. This research proposal is aimed specifically at providing the broad biomedical research community with the molecular information needed to better understand and more effectively treat PD.